Antibiotic resistance and virulence genes profiling of Vibrio cholerae and Vibrio mimicus isolates from some seafood collected at the aquatic environment and wet markets in Eastern Cape Province, South Africa

The current study determines the density of Vibrio spp. and isolates V. cholerae and Vibrio mimicus from fish-anatomical-sites, prawn, crab and mussel samples recovered from fish markets, freshwater and brackish water. Virulence and antibiotic resistance profiling of isolates were carried out using standard molecular and microbiology techniques. Vibrio spp. was detected in more than 90% of samples [134/144] and its density was significantly more in fish than in other samples. Vibrio. cholerae and V. mimicus were isolated in at least one sample of each sample type with higher isolation frequency in fish samples. All the V. cholerae isolates belong to non-O1/non-O139 serogroup. One or more V. cholerae isolates exhibited intermediate or resistance against each of the eighteen panels of antibiotics used but 100% of the V. mimicus were susceptible to amikacin, gentamycin and chloramphenicol. Vibrio cholerae exhibited relatively high resistance against polymyxin, ampicillin and amoxicillin/clavulanate while V. mimicus isolates exhibited relatively high resistance against nitrofurantoin, ampicillin and polymixin. The multiple-antibiotic-resistance-index [MARI] for isolates ranges between 0 and 0.67 and 48% of the isolates have MARI that is >0.2 while 55% of the isolates exhibit MultiDrug Resistance Phenotypes. The percentage detection of acc, ant, drf18, sul1, mcr-1, blasvh, blaoxa, blatem, blaoxa48, gyrA, gyrB and parC resistance-associated genes were 2%, 9%, 14%, 7%, 2%, 25%, 7%, 2%, 2%, 32%, 25% and 27% respectively while that for virulence-associated genes in increasing other was ace [2%], tcp [11%], vpi [16%], ompU [34%], toxR [43%], rtxC [70%], rtxA [73%] and hyla [77%]. The study confirmed the potential of environmental non-O1/non-O139 V. cholerae and V. mimicus to cause cholera-like infection and other vibriosis which could be difficult to manage with commonly recommended antibiotics. Thus, regular monitoring of the environment to create necessary awareness for this kind of pathogens is important in the interest of public health.


Introduction
water resources during a reconnaissance visit. The arrangement was made with them such that samples were recovered in our presence and immediately transferred into a sterile zip-lock bag. We also arranged with the fish market owners such that samples were purchased as soon as they got back from their fishing trip. Samples were purchased whole except in a few instances when fish heads and intestines have been removed by fishermen before returning from fishing trips. After purchase, samples were transported to the laboratory on ice packs in cooler boxes within six hours of collection. the line of streak coupled with Gram-staining. The presumptive isolates were confirmed as Vibrio spp. using the vibrio genus-specific primers and confirmed isolates were afterwards delineated into V. cholerae and V. mimicus using species-specific primers in a 25 μl PCR reaction. Furthermore, V. choleae isolates were screened for the presence of O1 and O139 strains using specific primers that targeted rfb regions responsible for "O" antigen biosynthesis [36,37]. The details of the primers and amplification conditions are given in S1 Table.

MARI for an isolate ¼ a=b ð1Þ
where a = number of antibiotics an isolate exhibited resistant against and b = the total number of antibiotics tested against the isolate. Multiple antibiotics resistance phenotypes (MARP) i.e. isolates that exhibited resistance against more than one antibiotic and multidrug antibiotics resistance phenotypes (MDRP) i.e. isolates that exhibited resistance against at least one antibiotic in three or more classes of antibiotics that were used for this study [45]. Isolates based on their phenotypic resistance profile were tested for the presence of some antibiotic resistance genes [cmlA, cat, flor, aac, ant, aphA, drf18, sul1, sul 2, mcr-1, blas shv , bla oxa , bla tem , bla vim , bla kpc , bla ndm , bla imp , bla oxa48 , gyrA, gyrB, parc, sxt] in a 25 μl PCR reaction.
Also, to understand the pathogenicity of the confirmed isolates, they were tested for the presence of eleven virulence determinants (details in S1 Table) in a 25 μl PCR reaction. The virulence genes selected are those that have been implicated in cholera and vibriosis caused by V. cholerae and V. mimicus. The multi-virulence gene indexes (MVGI) and the multi-resistance gene indexes of the isolates were also determined using a method reported elsewhere [46]. The antibiotics-resistant genes targeted, primers details and thermal conditions used for all PCR reactions are given in S1 Table.

Statistics
The Vibrio spp. mean density, MARI, MRGI, MVGI and MDRP was subjected to a normality test using Kolmogorov-Smirnov and Shapiro-Wilk test to determine whether to use a parametric or non-parametric test. Afterwards, an appropriate statistical test was employed to compare the Vibrio spp. density across sample types while the relationship between MARI, MVGI, MRGI and MDRP was determined using an appropriate correlation test. Correlation analysis between MARI, MVGI, MRGI and MDRP was carried out to observe the type of relationship that exists between phenotypic resistance, prevalence of virulence determinants, prevalence of resistance genes and prevalence of multidrug-resistant phenotypes. The differences between the magnitude of antibiotics resistance exhibited [using MARI as the corresponding variable], prevalence of virulence determinants (using MVGI as the corresponding variable) and prevalence of antibiotics resistance genes (using MRGI as the corresponding variable) across the Vibrio spp. (V. cholera and V. mimcicus) isolates, sampling sites, sample types, sample sources and sample class were also statistically determined. The significant level was set at <0.05. SPSS version 25 was used for the statistical analysis.

Vibrio spp. density and distribution of V. cholerae and V. mimicus in samples
The mean Vibrio spp. density across sample types is given in Fig 1 with gill having the highest mean density while mud prawn has the lowest. The density of Vibrio spp. for each sample type and site is given in Fig 1 and Table 1. A total of 29 fish samples which resulted in 29 flesh, 24 fins, 26 gills and 18 intestine samples were recovered from the fish markets and surface water. The crab, mud prawn and mussel samples collected from surface water were 24, 9 and 14 respectively. Of the 17 samples recovered from the fish markets, 41% were positive for one or both V. cholerae and V. mimicus. On the other hand, 25% (3/12) of the fish samples recovered from the surface water were positive for the two Vibrio species. At least one of the anatomical sites of all the fish samples was positive for Vibrio spp. however, Vibrio spp. was not isolated in 3 fins, 4 flesh, 2 intestine and one gill samples. Only 27.59% [8/29]   https://doi.org/10.1371/journal.pone.0290356.g001 IN = 2]) were confirmed as V. cholerae and V. mimicus respectively. Given the above, the prevalence of V. cholera among the population of confirmed Vibrio spp. from the flesh, fin, gill and intestine samples were 5.88%, 8.60%, 2.42% and 3.06% respectively while the prevalence of V. mimicus was 0%, 0%, 1.61% and 2.04% respectively. Seventy-four presumptive isolates were recovered from crab samples out of which 68% (51/75) were confirmed as Vibrio spp. while approximately 11% (8/75) and 3% (2/75) isolates of the confirmed Vibrio spp. were V. cholerae and V. mimicus respectively. The presumptive isolates from mud prawn samples were fifty out of which 76% (38/50) were confirmed as Vibrio spp. An 8% (3/38) and 0% (0/38) of the population of the confirmed Vibrio spp. were V. cholerae and V. mimicus respectively. The presumptive isolates recovered from mussel samples were 211 of which 92% (195/211) were confirmed as members of the vibrio genus. Of those confirmed, 1% (2/195) and 2% (4/195) were confirmed as V. cholerae and V. mimicus respectively. A total of 33 and 9 V. cholerae and V. mimicus respectively were recovered from samples and most of the isolates 59% (26/44) were recovered from fish anatomical sites. All the V. cholerae were confirmed to belong to non-O1/non-O139 serogroup.

Resistance-associated genes, virulence-associated genes and phenotypic resistance combinations detected in isolates
The highest number of virulence genes detected concurrently in a V. cholerae isolate was 6 and this is found in just one isolate ( Table 4). The highest number of the virulence determinants detected simultaneously in a singular V. mimicus isolate per time was four and this was observed in three V. mimicus isolates ( Table 4). The combinations of virulence genes, antibiotic resistance genes and phenotypic resistance observed are given in Table 4. A total of fourteen different combinations of the virulence genes targeted were detected. Only one virulence combination type was common to both V. cholerae and V. mimicus isolates while nine combinations were only found among the V. cholera population, four were found only among V. mimicus population. Eleven resistance gene profiles were observed, one profile was common to both V. cholerae and V. mimicus, one was only found among V. mimicus population and nine among only the V. cholera isolates. The highest resistance genes detected concurrently among V. cholerae isolate were five and two for V. mimicus. Twenty-one resistance phenotypes were detected of which one was common to the two Vibrio spp. while sixteen were found among V. cholerae isolates only and four among the V. mimicus population only. The highest concurrent phenotypic resistance detected in a V. cholerae isolate was against ten panels of antibiotics while that for V. mimicus was against thirteen. The distribution of resistance genes, phenotypic resistance and virulence genes combinations (profiles) among V. cholerae and V. mimicus populations is given in S3 Table while the dynamics of resistance for sampling sites, sample types, sources of samples and classes of samples using MARI, MARP AND MDRP as indices is given in S4 Table.

Statistical analysis
The variables (Vibrio spp. mean density, MVGI, MRGI and MARI) failed Kolmogorov-Smirnov and Shapiro-Wilk tests and thus tools for non-parametric data were used for statistical analysis. Welch ANOVA test result revealed a significant difference in Vibrio spp. density across sample types F [6,46.37] = 7.62 P < 0.001. Game-Howell post Hoc analysis result given in Table 5 indicated that Vibrio species. density in gill, fin and flesh samples were significantly more than that in mud prawn and crab samples. Other comparisons of density across sample types were not significantly different. Spearman rho correlation analysis carried out determined the relationship between MARI, MVGI, MDRP and MRGI because the four variables failed Kolmogorou-Smirnov and Shapiro-Wilk tests and for the same reason, the Wilcoxon test was used to compare the differences between the three variables (MARI, MVGI and MRGI) across Vibrio spp. while Welch ANOVA and Game-Howell post hoc analysis was used to compare the three variables across sampling sites, sample types, sample sources and sample classes. ANOVA was adopted for the interpretation where Welch tests cannot be performed due to a group having a variance of zero. The result of the correlation test (

Vibrio spp. density and the prevalence of V. cholerae and V. mimicus in samples
The density of Vibrio spp. differs numerically across anatomical sites of fish and other aquatic animals studied. The mean density of the Vibrio spp. in the fish anatomical sites except intestinal samples was significantly more than that of the crab and mussel samples. The gill sample has the highest average Vibrio spp. density and this is in concordance with an earlier study [53,54]. The relatively high occurrence of V. cholera and V. mimicus in fish samples when compared to other samples supports earlier studies that reported that fish is one of the main reservoirs of V. cholera and V. mimicus in the aquatic environment. [1,20,[55][56][57][58]. The low Cholera-causing V. cholerae serotypes (O1 and O139) are not commonly isolated from seafood as observed in this study nevertheless, the non-O1/non-O139 serogroups isolated have been implicated in human and aquatic animal infections outbreaks [60][61][62][63][64]. The relatively high prevalence of V. cholerae and V. mimicus in market samples than in surface water samples suggests a higher risk of contracting non-O1/non-O139 V. cholera and V. mimicus infections at the fish market in Eastern Cape than at the surface water. As relayed by the owners, the fish sold at the fish markets used for this study are from outside Eastern Cape Province. This calls for a more stringent microbial examination of fish products imported into the province.

The complexity and diversity of virulence determinants mix in non-O1 and non-O139 V. cholerae and V. mimicus isolates and implication
The current study shows the potential complexity and variation that could be observed in the virulence determinant profile of non-O1/non-O139 strains isolated from a similar geographical location. Horizontal gene transfers and serotype conversion has been linked to this observation. All four tcp + non-O1/non-O139 isolates were rtx + while two were toxR + . One of the toxR + isolates was hylA El Tor positive while the other was positive for vpi.
The ace gene, a core gene of the CTX elements which encodes enterotoxin detected in one of the isolates, is rarely found in non-O1/non-O139 strains but when detected, it is as a result of serogroup conversion of non-pathogenic environmental strains through horizontal gene transfer mechanisms to pathogenic ones [65]. The tcpA gene which is another core CTX element that encode the major structural unit of toxin-coregulated pilus detected in this study is believed to be a specific O1 and O139 serogroup virulence gene. However, the possibility of the acquisition of the tcp A gene by non-O1/non-O139 via horizontal gene transfer has been posited [66] and this explains its detection in the current study and an earlier study.
The VPI gene detected in 17% of the isolates in this study is an essential virulence gene cluster that enhances the colonization of the human intestine as well as the activity of the infection receptor site for most toxigenic Vibrio strains [67]. The toxin coregulated pilus [TCP], an essential colonization factor, is encoded within the VPI and the highland also encodes several virulence regulators and sialic acid utilization genes [68,69]. All the vpi-positive isolates in our study harboured the two rtx, toxR, hylA El Tor and one of them tested positive for tcpA gene as earlier reported [70]. On the other hand, tcpA + , toxR + vpistrains were detected and this is in line with studies that reported Vibrio cholerae strain that is positive for cholera toxin but negative for vibrio pathogenicity highland [71,72] and Vibrio alginolyticus, V. cholerae O1, O139 and non-O1/non-O139 strains that are vpi negative but tcpA positive [70,73]. Genomic sequence and comparative studies have confirmed the inter-specific and intra-specific differences in the content of the pathogenicity highlands. Also, it has been reported that the three Vibrio cholera highlands can excise from the chromosome and circular intermediates and VPI can be transferred from vpi+ strain to vpi-strain [74][75][76][77].
As usually reported, OmpU + V. cholerae isolates are also ToxR + since ToxR is needed for the regulation of OmPU gene [78][79][80][81][82]. However, only 61.5% of the OmpU + of non-O1/non-O139 isolates were ToxR + and the two OmpU + V. mimicus also lack the ToxR gene in this study. OmpU -ToxRand OmpU -ToxR + V. cholerae isolates have been reported [78][79][80][81][82][83] but OmpU + ToxRstrains are here reported for the first time. We suspected deletion or outright absence of the ToxR gene in the OmpU + ToxRstrains since ToxR gene could be chromosomal or plasmid-borne and deletion/acquisition of the gene has been reported [84][85][86][87][88][89][90]. The OmpU gene can still be expressed in the presence of the ompT promoter and the absence ToxR gene [88]. Although we did not target ToxT gene in this study the aforementioned explained the possibility of OmpU + and ToxRgenotypes in Vibrio species that will express OmpU in the absence of ToxR gene. None of the eleven virulence-associated genes was detected in 20% of the V. cholerae isolates in this study. The non-O1/non-O139 strain that lacks key structural and regulatory pathogenicity genes have been reported [65].
Vibrio cholerae virulence genes have been reported in other human vibrio pathogens such as V. alginolyticus and V. mimicus [65,70]. All the virulence-associated genes detected in the V. cholerae isolates in this study were also detected in V. mimicus isolates except for ToxR and ace genes. The study carried out by [8] on the genomic comparison between environmental V. mimicus and clinical isolates concluded that the genes [ctx, ace, zot and tcp] that is related to intestinal infections are common only with clinical V. mimicus isolates. This explains in part our result on the prevalence of the virulence genes targeted in the V. mimicus isolates. Furthermore, the none detection of the ToxR gene even in the OmpU + V. mimicus could be explained as earlier discussed for the V. cholerae. Nevertheless, none of the V. mimicus could be called avirulent because they all harboured at list one of the virulence determinant targeted in this study. Even if none of the targeted virulent-associated genes is detected in V. mimicus isolates, the targeted vmh gene for the PCR confirmation of the V. mimicus isolates is a virulence determinant [91]. The gene encodes a heat-labile hemolysin, designated V. mimcus hemolysin [5,92] based on their findings suggested the gene as the most important virulence factor of the pathogen. The toxR, zot, ctx, VPI, ompU, rtx and hylA genes have been reported in both clinical and environmental V. mimicus isolates [4,65,93] however, [94] believed that the tcpA + V. mimicus strain they reported and which we also isolated in the present study is rare. Our literature search confirmed that the strain is scarce nevertheless, it has been reported by [65] before the report of [94]. The analysis of the result of the virulence determinants in this study and our literature search showed that the peculiarity of virulence determinants among vibrio genus members is gradually diminishing. This supports the position of earlier reports and our study that members of the vibrio genus are evolving in terms of virulence. This evolution might lead to the emergence of pathogenic Vibrio spp. that are more virulent than those earlier known.
Our findings in terms of the complex mix of virulence determinants found in non-O1 and non-O139 V. cholerae strains and recent studies on virulence determinants of non-O1 and non-O139 strains suggested that V. cholerae is undergoing an evolution that might lead to the emergence of highly virulent strains. Atypical El Tor strains that share characteristics of prototype classical, the El Tor biotypes that is not known to exist before 2002 and strains that cause more severe cholera outbreak has been reported [95][96][97][98][99][100][101][102]. Also, the emergence of SA-NAG V. cholerae strain that shares genomic [e.g. presence of rtxC, El Tor and the Classical hemolysin gene] and phenotypic attributes of O139 and O1 serogroups have been reported [28]. The detection of the repeat toxin [rtxC] gene which is one of the genomic characteristics that confirms V. cholerae isolates as of the El Tor biotypes in non-O1 and non-O139 in this study is another pointer to the possible evolution of new V. cholerae [98,[103][104][105]. Summarily, the PCR detection of El Tor hemolysin gene in 74%, TcpA El Tor in 11% and rtxC genes in 71% of the non-O1/non-O139 isolates analyzed in this study supports the possibility of the emergence of a typical pathogenic SA-NAG V. cholera strains that could be more virulence than the known pathogenic strains. This affirms the need for surveillance for non-O1/non-O139 in clinical and non-clinical environments. The need for a surveillance programme for all Vibrio spp. in the clinical and environmental settings has been emphasized by a study carried out in Germany [106]. The call for this kind of program is due to the non-specific spread of virulence determinants among the members of the genus as well as the ongoing global warming challenge which is predicted to cause an increase in the abundance of Vibrio spp. and their infections in the clinical and non-clinical environment.

Virulence determinants detected in non-O1 and non-O139 V. cholerae and V. mimicus isolates and their role in pathogenicity
Eight of the eleven virulence genes targeted in this study were detected and of the eight detected, toxR, rtx and hlyA ELTor virulence genes predominate as earlier reported [65]. The rtxA is the most detected of the two rtx genes. Similar to earlier studies, some of the isolates in this study possessed both rtxA and rtxC genes, while neither of the two genes was detected in a few of the isolates [107,108] [63,109].
The detection of rtxA and rtxC in the current study is at per with some earlier studies [28, 65, 108, 110] although a higher prevalence of rtxA and rtxC is recorded in the current study. The actin cross-linking repeats in the toxin gene cluster [rtx] lead to actin depolymerization when cross-linked with Hep-2 and this causes villi effacement, hemorrhagic colitis, and bloody diarrhoea as earlier reported [103,111]. This suggests the need to screen for the SA-NAG Vibrio cholerae in the diarrhoea cases when common etiological agents [e.g. Escherichia coli and Vibrio cholera O1 and O139 serogroups] are not isolated/detected. It is also needful to screen for this non-O1/non-O139 serogroup when poly-bacterial diarrhoea infection is suspected.
ToxR and HylA El Tor are the other two prevalence virulence-associated genes in this study. The prevalence of toxR [74.3%] and hylA [74.3%] in our study are similar to that of earlier studies [63,108] from clinical and environmental samples. These two genes are part of the five [hlyA, hlyU, hlx, toxR and attRS1] virulence-associated genes which were found in the Vibrio cholerae non-O1/non-O139 strains that cause cholera epidemics in Calcutta, India. Surprisingly, all the strains isolated from the epidemics lacked the core virulence determinants [ctx elements and tcp genes] of the toxigenic V. cholerae O1 and O139 [66]. The regulatory protein ToxR controls the expression of the virulence determinants in V. cholerae O1, O139 [66] while the product of hylA causes haemolysis of erythrocytes [112]. It has been posited that Vibrio cholerae non-O1/non-O139 could acquire tcpA and ctxA which could favour the emergence of novel toxigenic strains under the biological and physicochemical selective pressures of the aquatic environment [63]. The Outer Membrane Protein U [OmpU] gene is another virulence determinant detected in the non-O1/non-O139 strains in this study. This gene is an adherence factor that has been reported in V. cholerae, V. alginolyticus and V. mimicus and it is one of the genes that are responsible for pathogens ability to colonize the intestine [88,[113][114][115]. The OmpU and OmpT genes are called pore-forming proteins of the outer membrane and they are regulated by ToxR gene [88].

Vibrio cholerae.
The antibiotics susceptibility pattern observed in this study varies across Vibrio spp., sample types, source of isolates and sampling sites. The V. cholerae isolates were either susceptible or exhibited intermediate susceptibility to amikacin, gentamycin, ofloxacin and cefuroxime. Gentamycin and amikacin interfere with protein synthesis, ofloxacin affect DNA synthesis while cefuroxime interferes with cell wall synthesis. Earlier studies have shown that V. cholerae non-O1/non-O139 are generally susceptible to the four antibiotics [43, 61,63,108,[116][117][118][119][120]. To interpret other susceptibility/resistance patterns for the remaining panel of antibiotics, resistance to an antibiotic was referred to as low to medium when the population that exhibited resistance against it is < 50% [121] and high when the population is > = 50% [122]. Going by this interpretation criteria, the V. cholerae exhibited high resistance to polymyxin, amoxicillin/clavulanate and ampicillin that target the cell wall synthesis but low/ medium resistance to the other eleven panels of antibiotics. The eleven antibiotics that V. cholerae exhibited low to medium resistance against can be categorized into three. Less than 10% of the V. choloerae isolates exhibited resistance against the first category which is made up of imipenem, meropenem and cefuroxime; while 10-20% exhibited resistance against the second group which is made up of kanamycin, azithromycin, cefotaxime, chloramphenicol and ciprofloxacin. A greater than 20% but less than 35% of the V. cholerae isolates exhibited resistance against the third group [nalidixic, nitrofurantoin, cotrimoxazole and trimethoprim]. The high resistance exhibited against polymyxin B and ampicillin has been reported and this seems to be common among environmental isolates of V. cholerae non-O1/non-O139 strains [108,123] and O139 toxigenic and non-toxigenic serogroup [124]. The high resistance to polymyxin B and the high prevalence of hylAElTor virulence determinant confirms that most of our V. cholerae isolates belong to El Tor biotype while others might have lost the El Tor marker [polymyxin B resistance] or belong to classical biotype. Polymyxin B resistance is one of the markers for detecting the El Tor biotypes however, it has been reported that the EL Tor biotype can lose this marker [125]. The El Tor strains compare to the classical strains are more adapted and resilient to the environment, cause a higher infection-to-case ratio and have more asymptomatic carriers [95,126]. Some studies have reported low resistance of the SA-NAG V. cholerae against amoxicillin/clavulanate [117,127,128] however, other studies like ours have shown that different V. cholerae serotypes can exhibit high resistance against amoxicillin/clavulanate [129,130].
The antibiotics commonly recommended for the management of vibrio infections fall into four classes of antibiotics [tetracyclines, fluoroquinolones, macrolides, and trimethoprim/sulfamethoxazole] [133][134][135]. Of the four recommended, three were used in this study and the percentage of V. cholerae non-O1/non-O139 strains that exhibited resistance against members of the three classes was mostly more than what has been reported in similar studies.
Although the V. cholerae population in this study exhibited low resistance against kanamycin, meropenem, imipenem, ofloxacin and ciprofloxacin, a high percentage of them exhibited intermediate susceptibility to five antibiotics. This is worrisome because intermediate susceptibility for an antibiotic means a higher dose than recommended is needed for the antibiotic to be effective [136,137]. The concern stems from the fact that these antibiotics are categorized as critically important antimicrobials for human medicine [138]. The aforementioned suggests that V. cholerae isolates in this study have the potential to cause cholera-like and vibriosis that might be difficult to manage with commonly recommended and relatively inexpensive antibiotics at the usual dosage. Unfortunately, using higher doses and expensive antibiotics raises resistance, side-effects and economic concerns [139][140][141].
In light of the aforementioned and the low/medium resistance exhibited by the V. cholerae population against 80% of the concerned antibiotics, we opined that most of the V. cholerae isolates in this study have the potential to cause cholera-like and vibriosis that might be difficult to manage with commonly recommended and relatively inexpensive antibiotics.

Vibrio mimicus.
The phenotypic resistance profile of V. mimicus population was similar to that of V. cholerae except for view differences. The differences include: 1. No V. mimicsu isolate exhibited phenotypic resistance against imipenem, chloramphenicol and ciprofloxacin which V. cholerae population exhibited low/medium phenotypic resistance against, 2. the V. mimicus population exhibited low/medium resistance against cefuroxime which none of the V. cholerae exhibited resistance against, 3. V. mimicus population exhibited high resistance against nitrofurantoin to which V. cholerae exhibited low/medium resistance and, 4. V. mimicus isolates exhibited low/medium resistance against polymyxin B, amoxicillin/clavulanate and ampicillin while the V. cholerae isolates exhibited high resistance against the three antibiotics. Reports on the antibiotics resistance profile of V. mimicus isolates are limited in the literature however, few studies we come across during our literature search reported resistance patterns of the organism against all members of the panels of antibiotics used in this study except amikacin [20,55,93,[142][143][144]. Interestingly, all V. mimicus isolates in this study were susceptible to amikacin. The level of resistance observed against each member of the panel of antibiotics in this study was similar in some instances and differs in others to the earlier reports. High resistance against kanamycin and cotrimoxazole was reported by [142] but no resistance to cotrimoxazole and very low resistance to kanamycin was reported three years later [144]. The current study reports low resistance against the two antibiotics while Gxalo Yamanaka and Aziz, [1989]. This same scenario goes for other antibiotics used against V. mimicus in this study and it suggests that local antibiogram result should inform the choice of antibiotics for the management of V. mimicus infections. In the light of this and considering the intermediate susceptibility result, amikacin, gentamycin and ciprofloxacin are the three antibiotics that can be considered good choice for the management of V. mimicus infections in our study area although this is subject to other factors such as cost and recommendations of the regulatory bodies such as World Health Organization, Center for Disease Control and Prevention and local regulatory bodies in-charge of our study area.

Genomic bases for the phenotypic antibiotic resistance observed in V. cholerae and V. mimicus
A recent systematic review and meta-analysis [145] pointed out the paucity of information on the presence of resistance genes in Vibrio spp. The frequency of detection of the twenty-two antibiotics resistance determinants focused on in this study varies for V. cholerae and V. mimicus. Forty-three per cent of the V. cholerae isolates and twenty-two per cent of the V. mimicus isolates were positive for at least one of the virulence determinants targeted in this study. The sxt element which is a conjugative-transposon-like mobile gene element that encodes multiple antibiotic resistance [146] was not detected in this study and this is in line with an earlier report [123]. Phenotypic-genotypic/genotypic-phenotypic resistance discrepancy reported in earlier studies was observed in the current study [147,148]. Of the six resistance genes [cmlA, cat, flor, acc, ant and aphA] that confer resistance against protein synthesis inhibitors, cmlA, cat, flor that coffer resistance against chloramphenicol [149,150] were not detected. The acc and ant that confers resistance against aminoglycosides [amikacin, kanamycin and gentamycin] [151], were detected. However, all isolates that carry the aminoglycosides resistance genes were either phenotypically susceptible or exhibited intermediate susceptibility to the aminoglycosides while one of the isolates surprisingly exhibited resistance against chloramphenicol. The aphA that encodes kanamycin resistance [152] was not detected in any of the isolates yet five isolates exhibited phenotypic resistance against kanamycin. Eighty-nine per cent [16/18] of the isolates that exhibited resistance against antifolate drug carries one or both of the Drf18 and sul1 gene that encodes antifolate resistance.
The current study supports the few reports that recently detected mcr-1 gene in Vibrio specie [153][154][155]. One V. cholerae isolate was positive for mcr-1 gene which confers resistance to polymyxin E [colistin], polymyxin B and this gene can cause cross-resistance to other antimicrobials [156][157][158][159]. This isolate was only positive for one more of the targeted resistance determinants [gyrB] but interestingly, it is a multidrug-resistant isolate with MARI of 0.56. This observation could be due to the presence of mcr-1 gene that causes cross-resistance to other antimicrobial agents in the isolate. Some bacteria which include V. cholerae are typically resistant to polymyxin [160] however detection of the plasmid-mediated mcr-1 gene in them raises public health concerns in terms of the spread of colistin resistance [161,162] because colistin is one of the antibiotics of last resort [157].
The information on the prevalence of β-lactamases in non-O1/non-O139 Vibrio cholerae is limited in the literature and we could not find information on the prevalence of β-lactamases in V. mimicus. Of the eight β-lactamses [blashv, blaoxa, blatem, blavim, blakpc, blandm, blaimp and blaoxa48] targeted in this study, four [blashv, blaoxa, blatem, blaoxa48] were detected and of the four, blashv was the most detected while blaoxa48 was the least detected. All the four detected β-lactamses were found in at least one V. cholerae while only blashv and blatem were detected among the V. mimicus isolates. The blaoxa-1 gene was detected in three V. cholerae isolates and one of the three aslo carries blaoxa48. The co-existence of bacteria carrying variants of blaoxa genes within the same ecological niche as observed in this study has been reported [163]. We expected the V. cholerae isolate that was positive for both blaoxa-48 and blaoxa-1 resistance genes to exhibit phenotypic resistance against more members of the β-lactam antibiotics used in this study than the two that carry only the blaoxa-1 because the blaoxa-1 gene is easily deactivated by penems [164]. Also, we do not expect blaoxa-48 positive isolate to exhibit a high level phenotypic resistance against the penems since the blaoxa-48 enzyme only hydrolyses penicillins at a high level, broad-spectrum cephalosporins at a moderate level, carbapenems at a low level but not susceptible to β-lactamase inhibitors [165]. However, surprisingly, the two isolates that were negative for the blaoxa-48 like but positive for blaoxa-1 were resistant to all of the β-lactam antibiotics used in this study except imipenem but the blaoxa-48 and blaoxa-1 positive isolate exhibited resistance to ampicillin and amoxicillin/clavulanate but susceptible to imipenem, meropenem, cefotaxime and cefuroxime. The blashv gene detected in the two V. cholerae isolates that were positive for only blaoxa-1 but not detected in the third isolate that was positive for blaoxa-48 and blaoxa-1 explains this observation because the association of oxacillnases gene with extended-spectrum β-lactamases [ESBL] genes such as blashv often increases the level of resistance to carbapenems [166]. The blashv gene is the most detected of all the eight β-lactamases producing genes targeted in this study but isolates harbouring it exhibited varying patterns [six patterns detected as shown in Table 4] of phenotypic resistance to ampicillin, penicillin, amoxicillin/clavulanate, cefuroxime and cefotaxime. Going by the definition of extended β-lactamases, broad spectrum β-lactamases and penicillinases [167][168][169][170], we suggest that our isolates carry different variants of blashv gene which could belong to any of the 149 fully characterized variants recently reported [168] because we noticed that primer used to target blashv is not specific for a particular variant when be BLAST it using National Center for Biotechnology Information PRIMER-BLAST tool. Also, the variants could either belong to the two newly detected variants [blashv-230 with accession number NG 148673.1 detected in E. coli and blashv-231 with accession number NG_ 148674.1 detected in Klebsiella pneumonia] which were part of the hits returned by the PRI-MER-BLAST. The three isolates with blatem gene were resistant to at least one of the penicillin antibiotics used in this study. The blatem gene is one of the Class A β-lactamases that exhibit a broad substrate hydrolysis profile against penicillins, cephalosporins, and, for a few of the enzymes, carbapenems [171].
Two phenotypic resistance patterns against β-lactam antibiotics [cefotaxime R -cefuroxime S and amoxicillin/clavulanate R -ampicillin S ] observed in this study are strange because we expect a fourth-generation cephalosporin to be more active than third-generation cephalosporin and a penicillin/beta-lactamase inhibitor to be active than penicillin alone. Also, we did not come across these phenotypes in the literature. A research article report and a review [168,172] have shown that the blasshv-mediated phenotypic and genotypic resistance could be so complex to explain at times due to various factors. Summarily the factors as pointed out in literature are: 1. the balshv family is highly diverse and complex [a large number of allelic variants including extended-spectrum β-lactamases [ESBL], non-ESBL and several not classified variants exist] in nature, 2. β-lactamase produced in small quantity do not contribute significantly to antibiotic resistance and, 3. overproduction of a β-lactamases could mask the effect of other β-lactamases [168,[173][174][175][176][177][178]. Furthermore, Vibrio parahaemolyticus isolates that are more susceptible to a third-generation cephalosporin [ceftazidime] than fourth-generation cephalosporin [cefepime] have been reported [179]. Also, an unusual case of resistance to amoxicillin/clavulanate but susceptibility to ampicillin in Enterobacter cloacae isolate has been reported [180]. However, the mechanism[s] for this rather weird phenotypic resistance pattern remains unexplained in the literature.
The genomic basis for quinolone resistance has been linked to mutation at quinolone resistance-determining regions [QRDRs] of gyrA, gyrB and ParC genes [181]. This region was targeted in this study using primers that are specific for mutation at the QRDRs of the three genes. Unfortunately, we had challenges sequencing the resulting amplicons which is the usual practice. Nevertheless, our result was interpreted using the only four articles found in the National Library of Medicine of the NCIB database that used the same primers we used for their studies [182][183][184][185]. Their works showed that mutation at the QRDRs always results in phenotypic resistance to quinolones hence we suspect the occurrence of mutation at the QRDRs of the gyr and par genes of 88% [15/17] of our isolates that exhibited resistance against the quinolones. Active efflux pump or qnr gene that were not targeted in this study could be responsible for the phenotypic resistance against quinolones exhibited by the other 22% [2/ 17]. Active efflux pump or qnr genes have been linked to phenotypic resistance even in the absence of mutation in the gyr and par genes [185,186]. It was observed that some isolates harbour resistance genes without corresponding phenotypic resistance. For example, the four V. cholerae isolates that were acc and ant positive were susceptible to gentamycin. This kind of phenomenon in which bacterial isolates were susceptible to chloramphenicol despite carrying the chloramphenicol resistance gene has been reported [128].
We noticed a higher rate of phenotypic resistance and detection of resistance genes in isolates from fish samples compared to that from crab, mud prawn and mussel samples. This suggests that Vibrio spp. from fish are of more public health significance. We also observed phenotypic resistance in the absence of corresponding resistance genes and the presence of resistance determinants without corresponding phenotypic resistance in isolates. This suggests that our isolates could be engaging other antibiotics resistance mechanisms such as efflux pump [either singly or in synergy with resistance genes] to archive antibiotics resistance. The energy-dependent efflux of multiple antimicrobial agents from bacterial cells of the Vibrio spp. is a widely recognized resistance mechanism [187] and some of the specific efflux pumps found in vibrio are EmrD-3, VceB and VcrM Multidrug Efflux Pumps [188].

Conclusion
The virulence genes, resistance genes and phenotypic resistance heterogeneous profiles observed among our isolates [ Table 6] suggest that the three variables [sampling sites, sample types and sources of the samples] got contaminated with V. cholerae and V. mimicus from different sources. The group of isolates from these variables have different profiles for resistant genes, virulence genes and phenotypic antibiotics resistance. Interestingly, to corroborate the aforementioned, the virulence genes, resistance genes and phenotypic resistance profiles is unique for each of the Vibrio spp. isolates as shown in S3 Table. Also, the report on the heterogeneity of the virulence profile of enteropathogenic E. coli from different sources supports our opinion [189]. Microbial source tracking coupled with genomic relatedness study on the isolates is expected to unravel this observation in our future study.
Considering the virulence status of our isolates, the relatively high percentages of multidrug-resistant phenotypes [ , and the detection of resistance determinants in our isolates, we propose that the environmental sources of our isolates pose a public health risk in terms of the spread of antibiotics resistant strains of V cholerae non-O1/non-O139 and V. mimicus which could cause vibriosis or cholera-like infections that may be difficult to manage with commonly recommended antibiotics. Thus, the findings of this study support the need for prospective surveillance for antibiotic resistance among bacterial isolates from the environment for understanding and minimizing the spread of antibiotic resistance. The CDC recommends regular surveillance for antibiotic resistance among bacterial isolates from any environment as this is essential for understanding and minimizing the spread of resistance [190].